The identification and quantification of specific nucleic acid sequences has been an area of great interest in molecular biology over the past two decades. Expression profiles within the area of identification and quantification of specific nucleic acids is currently being intensely studied. The ability to identify and quantify certain nucleic acids and their products has advanced a broad range of disciplines, such as individualized medicine, and evaluation of drug resistance.
While much has been learned about various methods for identifying and quantifying specific nucleic acid sequences, these methods lack accuracy and precision for identifying small variations in nucleic acid sequences, resulting in a high level of false positives and an inability to broadly apply these methods. Methods for identifying target nucleic acids with specific single base polymorphisms have proven to be ineffective when these nucleic acids are present in a high background of other nucleic acids including nucleic acids with sequences similar to the target nucleic acids. Probe design has limited the number of different variable nucleic acids that can be identified at a time, and require controlled backgrounds of known nucleic acids. Probes associated with methods such as Real Time PCR for detecting particular nucleic acid variants including microRNAs (miRNAs) have also been problematic because of non-specific binding to RT primers and lack of sufficient sensitivity to the target sequences.
miRNAs are short RNA oligonucleotides of approximately 22 nucleotides that are involved in gene regulation. miRNAs regulate gene expression by targeting mRNAs for cleavage or translational repression. The role of miRNAs in the development and progression of disease has only recently become appreciated. Deregulated miRNA expression is implicated in onset and progression of different diseases including, but not limited to embryonic malformations and cancers.
As a result of their small size, miRNAs have been difficult to identify using standard methodologies. A limited number of miRNAs have been identified by extracting large quantities of RNA. miRNAs have also been identified that contribute to the presentation of visibly discernable phenotypes. Expression array data shows that miRNAs are expressed in different developmental stages or in different tissues.
Moreover, because of their potential broad use in treating and diagnosing different diseases, there is a need in the art (yet unmet) to develop methods of identification, isolation and also quatitation of miRNAs. The present invention addresses the need by disclosing efficient and sensitive methods and compositions for isolating and quantitating miRNAs from different samples, including those wherein there is only minimum amount of a starting material available.